Test strip

ABSTRACT

An improved disposable test strip for use in amperometric measurement of analytes in complex liquid media, such as blood, which has three or more electrodes has been developed. This strip is designed so that different electrical potentials can be maintained between a common pseudo reference/counter electrode and each of the other electrodes upon the imposition of a common potential by an amperometric meter. This capability is imparted to the test strip by providing different circuit resistances for each of these other electrodes. The test strip can be utilized to measure a single analyte such as glucose with a background compensation via a “dummy” electrode or it can be used to measure the concentration of multiple analytes.

The measurement of analytes such as glucose in complex liquid media suchas human blood by amperometric methods using disposable test strips hasbecome widely used and is currently employed in a number of commercialproducts. In certain configurations it is advantageous to improve thesignal to noise ratio by employing a three electrode system in which oneelectrode serves as a pseudo reference/counter electrode to establish areference potential. Typically this is a silver/silver chlorideelectrode. A second, working electrode is coated with an enzyme whichpromotes an oxidation or a reduction reaction with the intended analyteand a mediator which transfers electrons between the enzyme and theelectrode. The third “dummy” electrode is coated with the mediator butnot the enzyme and it provides a measure of the current which arisesfrom other than the oxidation reduction reaction involving the targetanalyte. An example of such a system is described in U.S. Pat. No.5,628,980 to Carter, et al. (incorporated by reference herein) and isutilized in the MediSense QID glucose meter.

The three electrode system provides a good way to isolate the currentwhich arises from the oxidation reduction reaction involving the targets analyte such as glucose but it also imposes a higher current load onthe pseudo reference/counter electrode. In some testing environmentssuch as glucose meters used by diabetics in their homes it isimpractical or impossible to pretreat the samples to remove possibleinterferants. Thus with home use glucose meters the diabetic simplyapplies a sample of whole blood. Whole blood typically contains a numberof electrochemically active species whose concentration may vary fromperson to person or even from sample to sample from the same individual.The dummy electrode provides a measure of current arising from thepresence of these interferants thus allowing a normalization whichremoves their contribution to the current measured at the workingelectrode. However, in such a three electrode configuration the currentseen by the pseudo reference/counter electrode includes contributionsfrom both the working electrode and the dummy electrode. Thus in somecases the pseudo reference/counter electrode sees a significantlygreater current than it would in a two electrode configuration.

The pseudo reference/counter electrode in such a configuration is, inact, serving two roles which can be inconsistent if the current it seesbecomes too great. It serves, on the one hand, to provide a constanthalf-cell potential, i.e. a reference potential and, on the other hand,it also serves as a counter electrode balancing the electron transferoccurring at the working and dummy electrodes. For instance, in atypical glucose meter,mediator is becoming oxidized at the working anddummy electrodes so a reduction reaction needs to occur at the pseudoreference/counter electrode to balance the electron transfer. With thetypical Ag/AgCl pseudo reference/counter electrode this involves thereduction of silver ions thus consuming (or reducing) silver chloride.If too much silver chloride is consumed the pseudo reference/counterelectrode can no longer serve its function of providing a source ofconstant half-cell potential. In other words, the potential differencebetween the two electrode reactions such as the oxidation of a mediatorat the working electrode and the reduction of silver at the pseudoreference/counter electrode will actually shift as the reactionproceeds.

One approach is to redesign the pseudo reference/counter electrode tohandle higher current loads without displaying a significant shift inhalf-cell potential. This would normally mean increasing the size orsilver concentration of the pseudo reference/counter electrode relativeto the working and dummy electrodes. It is difficult to further reducethe size of the working electrode because its size has already beenminimized. It is limited by the economically acceptable procedures forreproducibly manufacturing millions of such disposable test strips. Onthe other hand, increasing the size or silver concentration of thepseudo reference/counter electrode would significantly increase the costof such three electrode disposable strips because silver is the mostexpensive material used in the construction of such strips.

Therefore, there is a need for three electrode disposable test stripsfor use in amperometric systems whose cost is comparable to twoelectrode test strips and yet have pseudo reference/counter electrodeswith about the same stability as in the two electrode test strips.

It has been discovered that the current load on the pseudoreference/counter counter electrode in a disposable test strip for usein amperometric measurements with a three electrode system can bedecreased and therefore its half cell potential better stabilized byincreasing the resistance of the dummy electrode. This allows threeelectrode test strips to give better performance without changing theoperating characteristics of the meters in which they are used.

Increasing the resistance of the dummy electrode not only reduces thetotal current passing through the pseudo reference/counter electrode butit also changes the potential at the dummy electrode's interface withthe sample. Thus it is possible to have a three electrode system whichcan simultaneously measure the concentration of two analytes. Theeffective potential at the “dummy” electrode with the higher totalresistance can be adjusted to be too low to effect an oxidationreduction reaction indicative of the concentration of one of the twotarget analytes.

It is preferred to have the resistance of the dummy electrode be atleast 1000 ohms greater than that of the working electrode and it isespecially preferred that the resistance differential be at least about4000 ohms.

It is also preferred that the resistance of the dummy electrode beincreased by putting a resistance in series with the active electrodesurface of this electrode. Thus both the area and nature of the activesurface of the dummy electrode are kept similar or identical to that ofthe working electrode. This can readily be achieved by increasing theresistance of the conductive track which connects the active electrodesurface to the meter which applies the potential and measures theresulting current. In the typical disposable strip for amperometricanalyte measurement three electrode surfaces are present on one end ofan elongated flat strip and three contact pads, one for each of theelectrode surfaces, are present on the other end of the strip. Eachelectrode surface is connected to its contact pad by a conductive track.The contact pads serve as the means to establish electrical contactbetween the strip and the meter which applies the potential and measuresthe resultant current. The conductive tracks are typically covered by aninsulating layer to prevent any short circuits between them.

It is particularly preferred to increase the resistance of theconductive track of the dummy electrode by narrowing its width. If thisconductive track is made of the same material as the working electrode'sconductive track and has about the same thickness as the conductivetrack of the working electrode it will have a higher resistance. Such amechanism of increasing resistance is particularly easy to implement inmass manufacturing.

An example of the present invention will be described in accordance withthe accompanying drawings, in which:

FIGS. 1a and 1 b are schematic diagrams depicting the conductive layersof electrodes of disposable test strips having dummy/second workingelectrodes with narrowed conductive layers;

FIG. 2 is a schematic diagram depicting the conductive layers ofelectrodes of a control disposable test strip;

FIG. 3 is an exploded view of a disposable test strip;

FIG. 4 is a perspective view of the assembled strip of FIG. 3; and

FIG. 5 is a series of plots of current in microamps versus time inseconds for a working electrode subjected to an initial potential of 400milliVolts in the presence of a glucose containing sample for variousdummy electrode configurations.

The three electrode disposable test strip for the amperometricmeasurement of analytes in complex liquid media is optimized to improvethe signal to noise ratio without imposing an excessive current load onthe reference/counter electrode by increasing the resistance of thedummy electrode, i.e. the electrode which carries the electrochemicalmediator also utilized at the working electrode but which has no enzymeor other reactant selected to engage the analyte in an oxidationreduction reaction. A typical environment for the application of thisconcept is the three electrode test strip described in U.S. Pat. No.5,628,890 for the determination of glucose in whole blood samples.

Such a test strip is typically constructed of an elongated strip of arigid electrically non-conducting material such as plastic. Suitableplastics include PVC, polycarbonate or polyester. Three conductivetracks are laid on this strip so as to establish independent conductivepaths from one end to the other. Each track terminates at the endadapted to be proximate to the meter used to apply electrical potentialand measure the resulting currents with a contact pad that interfaceswith the meter. At the distal end of the strip each track terminates inan electrode adapted to contact the complex liquid medium which carriesthe analyte to be measured. A typical medium is whole human blood and atypical analyte is glucose.

The working electrode is a pad which is coated with both a substancedesigned to engage the target analyte in an oxidation-reduction reactionand a mediator adapted to transfer electrons between the pad and theoxidation reduction reaction. A typical substance is an enzyme adaptedto promote the oxidation of glucose, such as glucose oxidase, and themediator is a compound which readily transfers electrons from theoxidation reduction reaction to the pad, such as a ferrocene derivative.

The “dummy” electrode is a pad which preferably has the same surfacearea as the working electrode and is coated with the same amount of thesame mediator as the working electrode. The concept is to provide anenvironment in the immediate vicinity of this “dummy” electrode which isessentially identical to that of the working electrode except for thesubstance, typically an enzyme, adapted to react with the targetanalyte. Then the spurious electrochemical reactions which might occurat the working electrode giving rise to noise are just as likely tooccur at the “dummy” electrode. Thus the signal arising from suchspurious reactions can be determined by measurement at the “dummy”electrode and subtracted from the total signal measured at the workingelectrode. This provides an improved signal to noise ratio.

The pseudo reference/counter electrode is a pad with a material such assilver/silver chloride which has both the oxidized and reduced form of aspecies to provide an essentially constant half-cell potential. So longas the relative proportions of the reduced and oxidized form of thisspecies such as silver and silver chloride are not substantially changedthe half-cell potential of this electrochemical couple will remainrelatively constant. This facilitates being able to maintain a knownconstant oxidation or reduction potential at the working electrode. Thisallows a production batch of disposable test strips to have a commoncalibration.

In the typical situation the disposable strips are utilized with a meterwhich functions to correlate the amount of current observed upon theapplication of an external potential to the contact pads of thedisposable strip to the amount of analyte present. This meter isdesigned to assume certain electrical characteristics will be observedupon the application of this external potential. One such assumption isthat the amount of current observed will decrease monotonically withtime. If the current does not decay in the expected manner the meter isprogrammed to abort the test. If the half-cell potential of the pseudoreference/counter electrode such as a silver/silver chloride electrodeshifts the current characteristics may indeed fail to meet theexpectations programmed into the meter causing an aborted test.

For example the half-cell potential of the silver/silver chlorideelectrode will shift if the proportion of silver to silver chloride ischanged. As current flows through this electrode silver is eitherreduced or oxidized, depending on the nature of the reaction occurringat the working electrode In the typical meter for sensing glucoseconcentration glucose is oxidized at the working electrode reducing themediator. The mediator then transfers the electron or electrons it hasgained in this reduction reaction to its electrode pad. These electronsare then taken up at the pseudo reference/counter electrode. In thetypical case this is a silver/silver chloride electrode and theelectrons are taken up by the reduction of silver ions transformingsilver chloride to silver metal.

If a sufficient amount of current passes through such a pseudoreference/counter electrode the proportion of silver to silver chloridewill change enough to cause a noticeable change in the half-cellpotential of this electrode. If this change becomes large enough thecurrent at the working electrode may no longer decay monotonically. Thisin turn will cause the meter to sense an error condition and abort thetest.

The current at the working electrode arises from the oxidation reductionreaction involving the target analyte and the subsequent transfer ofelectrons by the mediator. In the typical glucose meter glucose isoxidized by glucose oxidase and the mediator, for instance a ferrocenederivative, then transfers the electrons liberated by the oxidation ofthe glucose to its electrode pad. In detail the glucose oxidase becomesreduced by oxidizing the glucose in the sample which is exposed to thedisposable test strip and then is reoxidized by reducing the mediator.The mediator in turn becomes reoxidized by transferring electronsthrough its electrode pad to the circuit with the pseudoreference/counter electrode. Normally the current arising from thistransfer decays monotonically in accordance with the Cottrell equationas the mediator in reasonable diffusion distance to the electrode padwhich was reduced by reaction with glucose oxidase is reoxidized.However, this behavior is dependent upon the potential at the workingelectrode being held at or above a certain potential relative to thepseudo reference/counter electrode. If the potential at this pseudoreference/counter electrode shifts, the behavior at the workingelectrode may no longer follow this pattern.

The disposable strips are typically designed so that the pseudoreference/counter electrode does not undergo such a potential shift. Forinstance this electrode can be made large enough that the currentgenerated by the analyte concentrations typically encountered does notconsume enough silver ions to cause such a shift.

The use of a third, “dummy” electrode, however, imposes an additionalcurrent load on the pseudo reference/counter electrode. In the typicalglucose meter where an oxidation reaction occurs at the workingelectrode, the reduction reaction occurring at the pseudoreference/counter electrode must balance not only the oxidation reactionat the working electrode but also any oxidation reaction occurring atthe “dummy” electrode. This additional burden may be sufficient to shiftthe half-cell potential of the pseudo reference/counter electrode out ofits design range.

This is a particular problem in glucose meters which utilize aninitially reduced mediator such as a ferrocene derivative. In such ameter there is an initial high current load as the mediator is oxidizedat both the working and “dummy” electrodes. If there is also a highlevel of glucose in the sample being tested, there will also be a fairlyhigh current load from the reoxidation of mediator initially reduced asa result of the oxidation of the glucose. The combined current load hasa tendency to adversely effect the half-cell potential of the pseudoreference/counter electrode.

The total current load on the pseudo reference/counter electrode can bereduced by increasing the resistance in the overall circuit. However, itis impractical to change the resistance in the circuit involving theworking electrode. The meters used with the disposable test strips ofpresent concern are calibrated to correlate the level of current in theworking electrode circuit after sometime period or over some fixed timeinterval after exposure of the test strip to the sample to theconcentration of target analyte. Then the meters are distributed to alarge number of users who expect to use the meters with the disposabletest strips for a number of years. Thus it is impractical to make anychange in such test strips which would require a corresponding change inthe meter with which they are used.

It has, however, been found that the resistance in the “dummy” electrodecircuit can be increased without adversely effecting the interactionbetween the disposable test strip and its meter. The function of the“dummy” electrode is to allow subtraction from the total signal orcurrent at the working electrode of that portion attributable tosuperious oxidation-reduction reactions with species in the complexliquid medium other than the target analyte. This subtraction is only ofconcern at the time or over the interval during which the current at theworking electrode is measured for correlation to the analyteconcentration. Typically such measurements are made after the resistanceof the overall system is comparatively high after most of the oxidationat the working electrode has already occurred. It has been discoveredthat at this point the difference in electrochemical environments at theworking and “dummy” electrodes is insufficient to adversely effect thefunction of the dummy electrode.

The relative difference in electrochemical environment between theworking electrode and a “dummy” electrode with added resistance doestend to decrease as a test cycle proceeds. As the mediator subject toreoxidation at the working electrode decreases the effective resistancein the working electrode circuit increases, i.e. there are few speciesto support electron transfer. Thus although there will always be a fixeddifference in resistance between the working and “dummy” electrodescircuits the percentage difference will decrease as the effectiveresistance in the working electrode circuit increases.

In an alternative embodiment, the three electrode arrangement is used tosimultaneously measure the concentration of two analytes. In this casethere are two working electrodes and one pseudo reference/counterelectrode. The first working electrode is designed to operate with afirst substance that engages one of the target analytes in an oxidationreduction reaction at a relatively low potential. The second workingelectrode is designed to operate with a second substance that engagesthe other target analyte in an oxidation reduction reaction only at ahigher potential. For ease in manufacturing both working electrodes aretypically coated with is both substances and appropriate mediators.However, the test strip is designed so that the second substance whichis coated on the first working electrode remains inactive. Inparticular, the electrical resistance in the circuit path from thecontact pad connected to the first working electrode through the firstworking electrode is significantly greatly than the electricalresistance in the circuit path from the contact pad connected to thesecond working electrode through the working electrode. Thus when acertain electrical potential is applied to the contact pads of bothelectrodes relative to the pseudo reference/counter electrode, theeffective potential at the first working electrode is less than that atthe second working electrode, some of the potential drop having beenexpended traversing the higher circuit resistance.

The two analyte embodiment is applied to the simultaneous measurement ofketones and glucose by utilizing an enzyme mediator system for theketones which operates at +200 mV and an enzyme mediator system for theglucose which operates at +400 mv. In particular, hydroxy butyratedehydrogenase (HBDH) with a nicotinamide adenine dinucleotide (NADH)cofactor and a 1,10-phenanthroline quinone (1,10 PQ) mediator is usedfor the ketones and glucose oxidase with a ferrocene derivative mediatoris used for the glucose.

The low operating potential of the HBDH/NADH/1, 10 PQ system is asignificant advantage for an analyte like ketones which has a limitedlinear response range. In the case of ketones a linear response istypically expected only over a range of between about 0 and 8 milliMolar. By operating at a low potential interference from other specieswhich might undergo an oxidation reduction reaction at a higherpotential is avoided. In other words, the probability that anotherchemical species in the sample might become oxidized and deliverelectrons to the first working electrode thus making a superiouscontribution to the current sensed at this electrode is minimized.

The potential at the first working electrode is adjusted so that uponthe application of a 400 mV potential between the second workingelectrode and the reference/counter electrode the potential between thisfirst working electrode and the reference/counter electrode is 200 mV.This adjustment is effected by increasing the resistance of the circuitpath involving this electrode relative to that involving the secondworking electrode by an appropriate amount in one of the ways discussedhereinabove.

The current sensed at the first working electrode is the result of theoxidation of ketones while that sensed at the second working electrodeis the result of the oxidation of both ketones and glucose. The amountof current at each electrode can then be employed in a simplesimultaneous equation to determine the concentration of ketones andglucose in the same sample.

It is, of course, possible to coat only the first working electrode withthe ketones sensitive chemistry and to coat only the second workingelectrode with only the glucose sensitive chemistry. This would beexpected to result in higher manufacturing costs. Typically thedisposable test strips are manufactured by a series of printing steps sothat applying different chemistries to each working electrode wouldrequire additional printing steps.

A particular application of the concept of a high resistance dummyelectrode to the measurement of glucose is illustrated in FIGS. 1through 5. In the strips illustrated, the working electrode and thedummy electrode each had a surface area of 6.612 square millimeterswhile the pseudo reference/counter electrode had a surface area of 4.18square millimeters. The conductive tracks which connect the contact padsto the electrode pads are in most cases 0.801 millimeters. In two casesthe conductive track associated with the dummy electrode was narrowed to0.510 millimeters and 0.305 millimeters, as illustrated in FIGS. 1a and1 b.

Two different conductive layer prints are illustrated in FIGS. 1a (TrackA) and 1 b (Track B). A control conductive layer print, in which theworking and dummy electrodes have the same resistance, is shown in FIG.2. Referring to FIGS. 1a, 1 b and 2, the electrode configuration on thesensor strips has three printed layers of electrically conducting carbonink 2. The layers define the positions of the pseudo reference/counterelectrode 4, the working electrode 5, the dummy electrode 5 a andelectrical contacts 3.

Referring to FIG. 2, working electrode 5 has a track width 16 that isequal to track width 16 a of dummy electrode 5 a. Equal track widths 16and 16 a give the working electrode and dummy electrode equalresistances. Referring to FIGS. 1a and 1 b, track widths 16 b and 16 cof dummy electrode 5 a are narrower than track width 16 a of the controlin FIG. 2. The conductive layer of dummy electrode 5 a is narrowed inorder to increase the resistance of the dummy electrode relative to theworking electrode resistance. Track width 16 c is smaller than trackwidth 16 b. Thus, the resistance of dummy electrode 5 a in Track A (FIG.1a) is greater than the resistance of dummy electrode 5 a in Track B(FIG. 1b).

The composition of the conductive layers can also affect the resistanceof the electrodes. Generally, the conductive layers of the electrodesare printed at the same time with the same ink. The conductive layerscan be printed with a low carbon-content ink or a high carbon-contentink. Low carbon-content had a carbon content of between 30 and 31 weightpercent and a resin content of between 7 and 9 weight percent. The highcarbon-content ink has a carbon content of between 42 and 45 weightpercent, and a resin content of between 7 and 9 weight percent.

A suitable electrode sensor strip is illustrated in FIGS. 3 and 4.Referring to FIGS. 3 and 4, the electrode support 1, an elongated stripof plastic material (e.g., PVC, polycarbonate, or polyester) supportsthree printed tracks of electrically conducting carbon ink 2. Theseprinted tracks define the positions of the pseudo reference/counterelectrode 4, of the working electrode 5, of the dummy electrode 5 a, andof the electrical contacts 3 that are inserted into an appropriatemeasurement device (not shown). The conductive layer of dummy electrode5 a is narrowed in order to increase the resistance of the dummyelectrode relative to the working electrode.

The elongated portions of the conductive tracks are each overlaid withsilver/silver chloride particle tracks 6 a and 6 b, with the enlargedexposed area overlying 4, and 6 b and 4 together forming the pseudoreference/counter electrode. The conductive track or layer for dummyelectrode 5 a is not overlaid with silver/silver chloride. This furtherincreases the resistance of the dummy electrode. The conductive tracksare further overlaid with a layer of hydrophobic electrically insulatingmaterial 7 that leaves exposed only the positions of the pseudoreference/counter electrode, the working electrode and the dummyelectrode, and the contact areas. This hydrophobic insulating materialprevents short circuits. Because this insulating material ishydrophobic, it can confine the sample to the exposed electrodes. Apreferred insulating material is available as POLYPLAST□ (Sericol Ltd.,Broadstairs, Kent, UK).

The working electrode working area 8 is formed from an ink that includesa mixture of an enzyme, a mediator, and a conductive material. The dummyelectrode working area is formed from ink that includes a mixture of amediator and a conductive material without enzyme. The respective inksare applied to the positions 5 and 5 a of carbon tracks 2 as discreteareas of fixed length. Alternatively, instead of an enzyme, electrodelayer 8 can contain a substrate catalytically reactive with an enzyme tobe assayed. The conductive material in a preferred embodiment includesparticulate carbon having the redox mediator adsorbed thereon.

A printing ink is formed as an aqueous solution of the conductor andadsorbed redox mediator. For the working electrode, it also includes theenzyme or, alternatively, a substrate. When the analyte to be measuredis blood glucose, the enzyme is preferably glucose oxidase, and theredox mediator is a ferrocene derivative.

The ink can be screen printed. The ink can include a polysaccharide(e.g., a guar gum or an alginate), a hydrolyzed gelatin, an enzymestabilizer (e.g., glutamate or trehalose), a film-forming polymer (e.g.,a polyvinyl alcohol), a conductive filler (e.g., carbon), a redoxmediator (e.g., ferrocene or a ferrocene derivative), a defoaming agent,a buffer, and an enzyme or a substrate. The ink printed on a dummyelectrode lacks the enzyme or the substrate.

The pseudo reference/counter electrode 6 b is situated relative to theworking electrode 8 and dummy electrode 8 a such that it is in anon-ideal position for efficient electrochemical function. Theelectrodes are arranged not to minimize the effect of the resistance ofthe solution on the overall resistance of the circuit (as isconventional). Positioning the pseudo reference/counter electrodedownstream of the working electrode has the advantage of preventingcompletion of a circuit (and thus detection of a response) before theworking electrode has been completely covered by sample.

The electrode area is overlaid by a fine grade mesh 9. This meshprotects the printed components from physical damage. It also helps thesample to wet the pseudo reference/counter electrode and workingelectrode by reducing the surface tension of the sample, therebyallowing it to spread evenly over the electrodes. Preferably, this meshlayer extends over the whole length of the sample path, between andincluding, the application point and the electrode area. Preferably,this mesh is constructed of finely woven nylon strands. Alternatively,any woven or non-woven material can be used, provided it does notocclude the surface of the electrode such that normal diffusion isobstructed. The thickness of the mesh is selected so that the resultingsample depth is sufficiently small to produce a high solutionresistance. Preferably, the fabric is not more than 70 μm in thickness.Preferably the mesh has a percent open area of about 40 to about 45%, amesh count of about 95 to about 115 per cm, a fiber diameter of about 20to about 40 μm, and a thickness of from about 40 to about 60 μm. Asuitable mesh is NY64 HC mesh, available from Sefar (formerly ZBF),CH-8803, Ruschlikon, Switzerland.

The mesh can be surfactant coated. This is only necessary if the meshmaterial itself is hydrophobic (for example, nylon or polyester). If ahydrophilic mesh is used, the surfactant coating can be omitted. Anysuitable surfactant can be used to coat the mesh, so long as it allowsadequate even spreading of the sample. A preferred surfactant is FC 170CFLUORAD^(□) fluorochemical surfactant (3M, St. Paul, Minn.). FLUORAD^(□)is a solution of a fluoroaliphatic oxyethylene adduct, lowerpolyethylene glycols, 1,4-dioxane, and water. A preferred surfactantloading for most applications is from about 15-20 μg/mg of mesh. Thepreferred surfactant loading will vary depending on the type of mesh andsurfactant used and the sample to be analyzed. It can be determinedempirically by observing flow of the sample through the mesh withdifferent levels of surfactant.

A second layer of coarser surfactant coated mesh 10 is applied over thefirst mesh. This second mesh layer controls the influx of the sample asit travels from the application point toward the pseudoreference/counter and working electrode areas by providing a space intowhich the displaced air within the sample transfer path can move as thesample moves preferentially along the lower fine grade mesh layer 9 andpartially in mesh layer 10. The spacing of the larger fibers of thesecondary mesh layer, perpendicular to the direction of sample flow,helps to control the sample flow by presenting repeated physicalbarriers to the movement of the sample as it travels through thetransfer path. The regular pattern of the mesh fibers ensures that thesample progresses in stages and that only samples with sufficient volumeto generate an accurate response are able to pass all the way along thepathway and reach the pseudo reference/counter electrode.

Preferably, mesh 10 is of a woven construction, so that it presents aregular repeating pattern of mesh fibers both perpendicular to andparallel to the longest aspect of the strip. Generally, the second meshlayer should be substantially thicker than the first mesh, with largerdiameter mesh fibers and larger apertures between them. The larger meshpreferably has a thickness of from 100 to 1000 μm, with a thickness offrom 100 to 150 μm being most preferred. A preferred mesh has a percentopen area of about 50 to about 55%, a mesh count of from about 45 toabout 55 per cm, and a fiber diameter of from about 55 to about 65 μm. Apreferred mesh is NY151 HC mesh, also available from Sefar, CH-8803,Rushchlikon, Switzerland.

Mesh 10 is also provided with a coating of a suitable surfactant (unlessthe mesh itself is hydrophilic). Preferably, it is the same surfactantas that on the first mesh layer. The loading of surfactant is lower onmesh 10 than on mesh 9, providing a further barrier to movement ofsample past the transverse fibers of mesh 10. In general, a loading of1-10 μg/mg of mesh is preferred.

The mesh layers 9 and 10 are held in place by layers of hydrophobicelectrically insulating ink 11. These layers can be applied by screenprinting the ink over a portion of the peripheries of the meshes.Together, the layers and mesh surround and define a suitable sampletransfer path 12 for the sample to travel from the application point atthe furthest end of the strip towards the working electrode and pseudoreference/counter electrode. The ink impregnates the mesh outside ofpath 12. The insulating material thus defines sample transfer path 12 bynot allowing sample to infiltrate the area of mesh covered by the layersof insulating material. A preferred insulating ink for impregnating themesh layers is SERICARD^(□) (Sericol, Ltd., Broadstairs, Kent, UK).

The upper part of the electrode is enclosed by a liquid/vaporimpermeable cover membrane 13. This can be a flexible tape made ofpolyester or similar material which includes a small aperture 14 toallow access of the applied sample to the underlying surfactant coatedmesh layers. The impermeable cover membrane encloses the exposed workingelectrode and pseudo reference/counter electrode. Thus, it maintains theavailable sample space over the electrodes at a fixed height which isequivalent to the thickness of both mesh layers 9 and 10. This ensuresthat the solution resistance is kept at a high level. Any samplethickness up to the maximum depth of the two mesh layers is adequate inthis respect. Aperture 14 is positioned overlying the furthest end ofthe open mesh area, remote from the pseudo reference/counter electrode 6b, such that the exposed area of mesh beneath the aperture can be usedas a point of access or application for the liquid sample to bemeasured. The aperture can be of any suitable size large enough to allowsufficient volume of sample to pass through to the mesh layers. Itshould not be so large as to expose any of the area of the electrodes.The aperture is formed in the cover membrane by any suitable method(e.g., die punching). The cover membrane is affixed to the strip along aspecific section, not including the electrodes, the sample transfer pathor application area, using a suitable method of adhesion. Preferablythis is achieved by coating the underside of a polyester tape with alayer of hot melt glue which is then heat welded to the electrodesurface. The hot melt glue layer is typically of a coating weightbetween 10-50 g/m², preferably from 20 to 30 g/m². Pressure sensitiveglues or other equivalent methods of adhesion may also be used. Careshould be taken when the tape is applied, the heat and pressure appliedto the cover membrane can melt the SERICARD^(□) and can cause it tosmear onto adjoining areas.

The upper surface of the cover membrane can also be usefully providedwith a layer of silicone or other hydrophobic coating which helps todrive the applied sample onto the portion of exposed surfactant coatedmesh at the application point and thus make the application of smallvolumes of sample much simpler.

In use, a disposable test strip of the invention is connected, viaelectrode contacts 3, to a meter (not shown). A sample is applied toaperture 14, and moves along the sample transfer path 12. The progressof the sample is sufficiently impeded by mesh layer 10 to allow thesample to form a uniform front rather than flowing non-uniformly. Air isdisplaced thorough the upper portion of mesh layer 10 to and throughaperture 14. The sample first covers working electrode 5 in itsentirety, and only then approaches and covers pseudo reference/counterelectrode 4. This completes the circuit and causes a response to bedetected by the measuring device.

The effect of increasing the resistance of a dummy electrode in a systemfor measuring glucose in a whole blood sample was electronicallymodeled. In particular, Medisense G2a disposable test strips whichutilize glucose oxidase and a ferrocene mediator were tested usingvenous blood spiked with glucose to a concentration of 15 mM. Theelectronics was used to simulate the effect of having a dummy electrodewith each of five added resistances from zero to infinity (no dummyelectrode). An initial potential relative to the pseudoreference/counter electrode of 400 mV was imposed on the workingelectrode and the current at the working electrode was monitored overtime. The results were reported in FIG. 5.

FIG. 5 illustrates that as the resistance increases so does the currentat the working electrode. This is an indirect indication that the halfcell potential of the pseudo reference/counter electrode is beingstabilized. In an ideal situation the current at the working electrodeshould be independent of the resistance of the dummy electrode andshould just depend upon the rate at which glucose is oxidized. However,in the real world the extra current load imposed on the pseudoreference/counter electrode by the dummy electrode does cause anobservable shift in the half cell potential of the pseudoreference/counter electrode. This in turn has an effect upon the currentobserved at the working electrode. As the potential difference betweenthe working and pseudo reference/counter electrodes decreases because ofthis shift so does the current at the working electrode.

In addition, under some conditions the current decay at the workingelectrode departs from the expected model. In particular, it is expectedthe current will decrease monotonicly with time and tend to exhibit thebehavior predicted by the Cottrell equation. However, under certainconditions when the dummy electrode is imposing a significant currentload on the pseudo reference/counter electrode the current at theworking electrode departs from classical behavior and may actuallyincrease with time over some short time period. This is clearlyillustrated in the lowest most curve of FIG. 5, which represents adisposable test strip in which there is no resistance differentialbetween the circuit path involving the working electrode and thatinvolving the dummy electrode.

The glucose meters with which the disposable test strips of presentconcern are typically used have electronic features designed to detectinvalid test results. One of these check features is a monitoring of thecurrent decay at the working electrode. If this decay is not monotonicthe meter will report an error condition and abort the test.

Thus increasing the resistance of the dummy electrode has been shown tobe effective in decreasing the likelihood of a non-monotonic currentdecay at the working electrode and the consequent abortion of a test.

We claim:
 1. A disposable test strip suitable for attachment to thesignal readout circuitry of a meter which performs an amperometric testto detect a current representative of the concentration of an analyte ina complex liquid medium comprising: (a) a working electrode whichcomprises an electrode pad coated with both a substance designed toengage said analyte in an oxidation-reduction reaction and a mediatorcompound which will transfer electrons between the oxidation-reductionreaction and the electrode pad; (b) a dummy electrode which comprises anelectrode pad which is coated with about the same amount of mediatorcompound as the working electrode but lacks the substance which engagesthe analyte in the oxidation-reduction reaction; (c) a pseudoreference/counter electrode which comprises an electrode pad coated witha material which contains both the oxidized and reduced form of achemical species which is designed to undergo a reduction or oxidationreaction to balance the opposite reaction at the working and dummyelectrodes; and (d) three conductive tracks, each of which extends froma contact pad adapted to interface with said readout circuitry to one ofthe electrode pads and which is in electrical contact with both itscontact pad and its electrode pad; wherein the electrical resistance inthe circuit path from the contact pad connected to the dummy electrodethrough the dummy electrode is significantly greater than the electricalresistance in the circuit path from the contact pad connected to theworking electrode through the working electrode.
 2. The disposable teststrip of claim 1 wherein the greater electrical resistance in the dummyelectrode circuit is provided by increasing the resistance of theconductive track connecting the dummy electrode to its contact pad. 3.The disposable test strip of claim 1, further comprising an elongatesupport having a substantially flat, planar surface arranged to bereleasably attached to the readout circuitry.
 4. The disposable teststrip of claim 3 wherein the three conductive tracks are created bycoating conductive particles on the elongated support.
 5. The disposabletest strip of claim 4 wherein the conductive particles comprise carbon.6. The disposable test strip of claim 4 wherein a greater electricalresistance is imparted to the conductive track connecting the dummyelectrode to its contact pad by using a smaller volume of conductiveparticles in this track as compared to that used in the conductive trackconnecting the working electrode to its contact pad.
 7. The disposabletest strip of claim 1 wherein the conductive track connecting the dummyelectrode to its contact pad is narrower than the conductive trackconnecting the working electrode to its contact pad.
 8. The disposabletest strip of claim 1 wherein the conductive track connecting the dummyelectrode to its contact pad is thinner than the conductive trackconnecting the working electrode to its contact pad.
 9. The disposabletest strip of claim 1 wherein the conductive track connecting the dummyelectrode to its contact pad has a different composition than theconductive track connecting the working electrode to its contact pad.10. The disposable test strip of claim 9 wherein both the conductivetrack connected to the dummy electrode and the conductive trackconnected to the working electrode are comprised of carbon particles butonly the latter conductive track is coated with silver.
 11. Thedisposable test strip of claim 1 wherein the conductive track connectingthe dummy electrode to its contact pad is longer than the conductivetrack connecting the working electrode to its contact pad.
 12. Thedisposable test strip of claim 1 wherein the analyte is glucose and thesubstance engaging the analyte in an oxidation reduction reaction is anenzyme.
 13. The disposable test strip of claim 12 wherein the enzyme isglucose oxidase.
 14. The disposable test strip of claim 1 wherein themediator is a ferrocene derivative.
 15. The disposable test strip ofclaim 1 wherein said pseudo reference/counter electrode comprises anelectrode pad coated with a mixture of silver and silver chloride. 16.The disposable test strip of claim 1 wherein the electrical resistancein said dummy electrode circuit is at least 1000 ohms greater than insaid working electrode circuit path.
 17. A disposable test stripsuitable for attachment to the signal readout circuitry of a meter whichperforms an amperometric test to detect currents representative of theconcentrations of multiple analytes in a liquid medium comprising: (a) afirst working electrode which comprises an electrode pad coated withboth a substance designed to engage one of the multiple analytes in anoxidation-reduction reaction at a first electrical potential differenceand a mediator compound which will transfer electrons between itsoxidation-reduction reaction and its electrode pad; (b) a second workingelectrode which comprises an electrode pad which is coated with both asubstance designed to engage another of the multiple analytes in anoxidation-reduction reaction at a second electrical potential differencewhich is significantly greater than said first electrical potentialdifference and another mediator compound which will transfer electronsbetween its oxidation-reduction reaction and its electrode pad; (c) apseudo reference/counter electrode which comprises an electrode padcoated with a material which contains both the oxidized and reduced formof a chemical species which is designed to undergo a reduction oroxidation reaction to balance the opposite reactions at the first andsecond working electrodes; and (d) three conductive tracks, each ofwhich extends from a contact pad intended to interface with said readoutcircuitry to one of the electrode pads and which is in electricalcontact with both its contact pad and its electrode pad; wherein theelectrical resistance in the circuit path from the contact pad connectedto the first working electrode through the first working electrode issignificantly greater than the electrical resistance in the circuit pathfrom the contact pad connected to the second working electrode throughthe second working electrode.
 18. The disposable test strip of claim 17wherein there are only two working electrodes.
 19. The disposable teststrip of claim 17 wherein the pseudo reference/counter electrodecomprises an electrode pad coated with a mixture of silver and silverchloride.
 20. The disposable test strip of claim 17 wherein the firstworking electrode comprises an enzyme system adapted to engage ketonesand a suitable mediator and the second working electrode comprises anenzyme suitable to engage glucose and a suitable mediator.
 21. Thedisposable test strip of claim 20 wherein the first working electrodecomprises a HBDH/NADH/1, 10 PQ system and the second working electrodecomprises a glucose oxidase and a ferrocene based mediator.
 22. Thedisposable test strip of claim 21 wherein the resistance in the firstworking electrode circuit is such that when a 400 mV potential existsbetween the second working electrode and the pseudo reference/counterelectrode there is a 200 mV potential between the first workingelectrode and the pseudo reference/counter electrode.